Nanobody Production

We have the Alpacas. We can make Nanobodies® for you.

About the Nanobody Project

We are now offering a single domain antibody (Nanobody®) production service. Nanobodies, in contrast to conventional antibodies which are made up of two heavy and two light chains with a molecular weight of ~150,000, are composed of only heavy chains. The heavy chain nanobody domains can be isolated as a small 15,000 Da single domain antibody, which retains the high affinity of conventional antibodies. Due to their small size nanobodies can be expressed in E. coli and their cDNAs manipulated in a variety of ways. Nanobodies are more heat stable than conventional antibodies and are stable over a wide pH range. These and other properties have made nanobodies increasingly popular as reagents for research, for use as therapeutics, and as reagents for diagnostics. 

Through resources provided by the National Institute of General Medical Sciences, the University of Kentucky’s COBRE Protein Core is now producing these single domain antibodies in alpacas for investigators as part of their fee for service structure. To generate the nanobodies, our facility immunizes alpacas with your protein of interest, amplifies the pool of nanobodies produced by the alpaca using PCR and generates a phagemid library expressing them in E. coli. The E. coli containing the nanobodies of interest are enriched by “panning” with your protein. Nanobody expressing E. coli are identified by PCR using in-house primers, and by sequencing. The nanobodies against your protein are confirmed by immunoprecipitation, expressed, and purified from E. coli. Using this protocol we have had a very high success rate. It should be noted that nanobodies produced in this manner are frequently conformation dependent. Limited success has been achieved making nanobodies to peptides or peptide conjugates. Thus, we do not use our alpacas to attempt to make nanobodies to peptides or peptide conjugates, just proteins. 


To produce a nanobody to your protein we need 3 mg of your purified protein in PBS or HBS and a gel showing the level of purity. There is a $250 non-refundable setup fee which will be applied to the final cost. We will deliver your nanobody usually within 3-4 months. We will ship you a sequenced cDNA that can be used for bacterial expression of your particular nobody as well as 1 mg of purified nanobody validated to bind to your protein. For investigators in IDEA states the cost of nanobody production will be subsidized by our COBRE grant. We typically identify more than one nanobody per antigen, and these can also be purchased. We can also conjugate your nanobody to fluorescent proteins (i.e. GFP), to Fc for immunoprecipitation, or other tags as desired. 

We will be producing nanobodies again in the Fall/Winter of 2018. For the Fall/Winter 2018 production cycle we will need the 3 mg of your purified protein by October 26, 2018 and a one sentence description of your anticipated use of the nanobody. The latter is required by our IACUC committee and will be kept confidential. The cost of our nanobody services is listed below. To order please fill out the nanobody order form and return via email to Christy Maddox (Christy.Maddox@uky.edu) or Lou Hersh (lhersh@uky.edu) by October 19th, 2018

Apply to Core

 

Suggested reading for nanobodies and their potential uses.

Exploiting Nanobodies' Singular Traits. Ingram JR, Schmidt FI, Ploegh HL. Annu Rev Immunol. 2018 Feb 28.

Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. Yamagata M, Sanes JR. Proc Natl Acad Sci U S A. 2018 Feb 27;115(9):2126-2131.

Editorial: Single-Domain Antibodies-Biology, Engineering and Emerging Applications. Henry KA, MacKenzie CR. Front Immunol. 2018 Jan 23;9:41.

Nanobodies as novel agents for cancer therapy. Revets H, De Baetselier P, Muyldermans S. Expert Opin Biol Ther. 2005 Jan;5(1):111-24. Review

Nanobodies and their potential applications. Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P, Vincke C, Muyldermans S.  Nanomedicine (Lond). 2013 Jun;8(6):1013-26.

No